Quantum Dots (QD) are quantum confined semiconductor nanoparticles. QDs exhibit luminescence properties when excited at a suitable wavelength and exhibit, in part, a size dependent emission wavelength as it is known in the art. Quantum Dots offer many advantages over traditional organic fluorescent dyes due to their unique properties such as 1) continuous absorbance and narrow emission bandwidth 2) ability to excite Quantum Dots emitting at different wavelengths simultaneously at a single excitation wavelength 3) ability to tune emission wavelength by the size of the semiconductor crystal and/or by the composition of the Quantum Dot in a broad spectral region 4) large absorbance cross-section and high molar absorptivity 5) long luminescent lifetime which allow longer analysis/detection time. Also, surface of QDs can be functionalized as hydrophilic or hydrophobic for suspension in aqueous or organic (oily) medium. Therefore, lately, quantum dots (QDs) are replacing organic fluorophores.
In the field of biotechnology and medicine, spectral window where quantum dots are excited and emitting is very critical. Best spectral window is usually accepted as 700-900 nm where the natural tissue has no or minimal absorbance and luminescence. This not only provides a better contrast in the imaging but also provides possibility for photons to penetrate or travel several centimeters in tissue, therefore allow relatively deeper tissue imaging.
An important handicap of Quantum Dots is the toxicity problem. Most common structures such as cadmium chalcogenides usually are excited at UV-range and emit in the visible region which is not a desired spectral window and these quantum dots are toxic. There are different approaches to solve these problems: One of which is the surface modification of the quantum dots with biocompatible materials. For example, a carbohydrate encapsulated quantum dot was disclosed in a patent application, WO 2005/093422 A2, in 2005. Another example to biological coatings is reported in a patent application, EP 1 868 938 A2, wherein Gluthathione encapsulated CdTe quantum dot is synthesized. Similarly, several core/shell or core/shell/shell type structures wherein the shell is again a chalcogenide with usually a broader band gap and preferentially free of Cd were also reported. (N. Chen et al. Biomaterials 33 (2012), 1238-1244)
Another limitation in this area is the challenge of synthesizing a good quality quantum dot. Good quality means, relatively monodisperse, with narrow omission bandwidth and having high quantum yield. Generally speaking, good quality quantum dots are prepared in organic solvents, from relatively toxic starting materials at high temperatures (200-400° C.). Therefore the resulting hydrophobic particles turn out to be unsuitable for biomedical applications and the surfaces of such QDs are modified with organic molecules which will allow suspension of these particles in aqueous medium. However, this process is time consuming, expensive and usually causes loss in luminescence properties and a shift in the luminescence wavelength and may also result in aggregation of particles. Aqueous synthesis of quantum dots is an alternative for the organic synthesis, but this approach most of the time results in quantum dots with broader size distribution, broader bandwidth of emission an poorer quantum yield. (M.-C. Castanon et al. Materails Letters, 2005; 59: 529)
Cationic quantum dots are promising nanomaterials in big logical applications especially in gene delivery studies— The most prominent cationic polymer is polyethyleneimine (PEI) for gene delivery. Polyethyleneimine (25 kDa branched) is accepted as gold standard in gene delivery applications because of high transfection efficiency. The polymer has been used to synthesize quantum dots to be used in biological applications. PEI coated CdS quantum dots were prepared with a broad (white-like) emission profile and quantum yields up to 60-70% at room temperature. However, these quantum dots emit in the visible region and have toxicity problem. Also most of the Ag2X. (X:S, Se and Te)-NIRQDs in literature were synthesized by organic, preparation methods. Examples to such studies are given in the following references; Du Y., et al. JACS 2010; 132:1470-1; Jiang P. et al., Chemistry of Materials 2011; 24:3-5; Sahu A. et al. JACS 2011; 133: 6509-12 and Yarema M. et al. ACS Nano. 2011; 5: 3758. Those quantum dots are emitting in the range of 700-1100 nm wavelength with very low quantum yields (up to 2%).
Recently, there are some reports on the synthesis of Ag2X-NIRQDs in aqueous medium Acar et al., prepared 2-mercaptopropionic acid coated Ag2S NIRQDs emitting in the 780-950 nm range with quantum yields up to 17% which is improved up to 39% upon aging. These Ag2S NIRQDs have the highest quantum yield reported until now (J. Mater. Chem., 2012, 22, 14674). Yang et al. reported Ag2S-BSA QDs synthesized in aqueous medium with emissions between 1050-1295 nm which is the second NIR range (Yan et al, Nanotechnology, 2013, 24, 055706). Glutathione stabilized Ag2S QDs were also prepared and reported with 0.96-1.97% quantum yield with emission between 960-1050 nm (Tan et al., ACS Applied Materials & Interfaces, 2013, 6, 18-23)
In a patent application WO 2012/163078 A1, a silver sulfide based quantum dot with a hydrophilic coating(s) is synthesized and claimed to have high fluorescence yield and stability, high biocompatibility and homogeneous size. And also a method of synthesizing this QD via a simple, easy to control, easy to implement route of synthesis is reported in this patent application. Although the synthesis method is claimed to be convenient, the defined method involves two step syntheses in which the first step produces organic soluble hydrophobic particles at 80-350° C. and the second step involves transfer of particles into aqueous phase through ligand exchange of the hydrophobic molecule with a hydrophilic one. As mentioned before, this method causes a shift of emission peak towards longer wavelengths and some broadening in the emission peak. Also, those particles have a crystal core size around 5 nm and luminesce around 1200 nm. This falls into second NIR window and expensive and low sensitivity InGaAs detector is required for the luminescence detector.
From this respect, it is apparent that there is no solution to all these obstacles in the prior art: the problems being appropriate emission window such as 700-900 nm, low quantum yields, cytotoxicity, transfection efficiency and mild conditions for the method of synthesis. In the literature there is no example to a method of synthesizing such quantum dot via an aqueous, one-step, low temperature route. Here, in this invention a novel cationic quantum dot which is capable of luminescence at the desired near-IR range at the wavelength of 700-900 nm with dramatically improved quantum yield and high transfection efficiency and low toxicity is claimed and also a method of synthesizing such quantum dot via a single step and aqueous reaction at low temperatures.
The technology behind this invention is the mixed coating of the quantum dots and the use of silver chalcogenide as nontoxic near-IR emitting quantum dot. With the mixed coating approach that is followed in this invention, a dense packing on the surface of the quantum dot is suggested therefore the problems related to defects that may exist resulting in non-radiative coupling events and low luminesce is eliminated. It had been demonstrated that mixed coating approach shows a synergistic effect on stabilization and reduction of the particle size. In the mixed coating approach, a small molecule preferentially a thiolated molecule and a macromolecule capable of binding to Silver-chalcogenide crystal surface was used as a coating material for the semiconductor crystal. Addition of small molecules next to polymeric coatings results in limiting the crystal growth and improvement of surface pasifization since they bind to surface strongly and more densely at sites left unpassified by the large polymeric coating. Therefore, mixed coatings are proved to produce smaller particles with better luminescence intensity compared to quantum dots coated only with polymeric coating. With this approach behind, aqueous cationic Near-IR emitting silver sulfide quantum dots (Cat-Ag2S-QD) with maximum emission at around 800-850 nm with quantum yields up to 150% with respect to LDS (LDS 798 Near-IR laser dye, quantum yield is 14% was reported by the producer) are synthesized at room temperature in aqueous medium. This luminescence window is suitable for detection with sensitive and cheap Si detectors. High quantum yields are highly advantages for improved penetration/imaging depth and signal/noise ratio and more efficient than high concentration (Won et al, Molecular Imaging, Vol 11, No 4, pp 338-352, 2012). Cytotoxicity studies done on several quantum dots (Cat-Ag2S-QDs) demonstrated the significant improvement of cytocompatibility as compared to 25 kDa polyethyleneimine which is accepted to be the gold standard for gene transfection. Also with the cell uptake studies it is demonstrated that the Cat-Ag2S-QDs are internalized by cells and may be used as optical probes in optical imaging. Also, in vitro studies demonstrated that Cat-Ag2S-QDs are capable of condensing and protecting GFP and the transfection efficiencies are enhanced as compared to PEI.